In plants, 4-hydroxypenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is a key enzyme in the biosynthesis of plastoquinones and tocopherols. 4-hydroxyphenylpyruvate acid (derived from chorismic acid via the shikimate pathway) is oxidized and decarboxylated by HPPD to yield homogentisic acid (Fiedler and Schultz, Dev. Plant Biol. 8:537, 1982; Fiedler et al., Planta 155:511, 1982). Subsequent polyprenylation and decarboxylation of homogentisic acid results in an array of plastoquinones and tocopherols.
In animals, HPPD is involved in tyrosine catabolism. A genetic deficiency in this pathway in humans and mice leads to hereditary tyrosinemia type 1. This disease can be treated by NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, an inhibitor of HPPD, which prevents the buildup of intermediates of tyrosine catabolism that are hepatotoxic (Ellis et al., Tox. and Appl. Pharm. 133:12, 1995).
Since plastoquinones and tocopherols are essential compounds for plants, inhibitors of this enzyme are potential herbicides. One class of HPPD inhibitors, the triketones, have recently been shown to possess herbicidal activity (Prisbylia et al., Brighton Crop Protection Conference: Weeds, British Crop Protection Council, Surrey, UK, pp 731-738, 1993; Schulz et al., FEBS Letts. 318:162, 1993). The corn-selective herbicide sulcotrione (2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione) causes strong bleaching in susceptible plants accompanied by a loss of carotenoids and chlorophyll with an increase in phytoene and tyrosine (Barta et al., Pest.Sci. 45:286, 1995; Soeda et al., Pestic.Biochem.Physiol. 29:35, 1987; Mayonado et al., Pestic.Biochem.Physiol. 35:138, 1989). Treatment of Lemna with sulcotrione severely inhibited growth and the herbicidal effect could be abolished with homogentisic acid. The partially purified enzyme extracted from maize was shown to be severely inhibited by sulcotrione with a calculated IC.sub.50 of 45 nM (Schulz et al., 1993, supra). Analysis of partially purified HPPD from barnyardgrass (Echinochloa crus-galli L.) showed sulcotrione to be a potent competitive inhibitor of the enzyme with a K.sub.i of 9.8 nM (Secor, Plant Physiol. 106:1429, 1994). Canadian Patent Application No. 2,116,421 describes the identification of HPPD inhibitors derived from 2-benzoylcyclohexamine 1,3-diones.
An albino mutant (psdl) isolated from a T-DNA tagged Arabidopsis population was originally selected by virtue of a severe pigment deficiency, which was thought to be due to a defect in carotenoid biosynthetic genes (Norris et al., Plant Cell 7:2139, 1995). When the albino psdl mutant was germinated on MS2 medium and subsequently transferred to MS2 medium supplemented with either 4-hydroxyphenylpyruvate (OHPP) or homogentisic acid (HGA), the plants greened on HGA but not OHPP. Further analysis of this mutant indicated that the defect causing the albino phenotype is not due to a mutation in a carotenoid biosynthesis enzyme directly, but rather results from a mutation in HPPD that prevents the biosynthesis of a plastoquinone essential for carotenoid biosynthesis.
Despite the importance of this pathway in plants, genes encoding the plant enzymes for plastoquinone and tocopherol biosynthesis have not previously been isolated. Thus, there is a need in the art for methods and compositions that provide HPPD genes, HPPD inhibitors useful as herbicides, and herbicide-resistant HPPD variants. The present inventors have isolated the gene encoding plant HPPD, have expressed it in E. coli, and have demonstrated that bacterially expressed plant HPPD is enzymatically active and that its enzymatic activity is inhibited by triketone herbicides.